Catechins Induce Oxidative Damage to Cellular and Isolated DNA through the Generation of Reactive Oxygen Species

Green tea catechins have antimutagenic and anticarcinogenic activities. On the other hand, several epidemiological studies have indicated significant positive relationship between green tea consumption and cancer. Catechins enhance colon carcinogenesis in rats initiated with chemical carcinogen. To clarify the mechanism underlying the potential carcinogenicity, we investigated the DNA-damaging ability of catechins in human cultured cells. Catechin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H₂O₂)-resistant cell line derived from HL-60. The catechin-induced formation of 8-oxodG in HL-60 cells significantly decreased by bathocuproine. Furthermore, we investigated DNA damage and its site-specificity induced by catechins, using ₃₂P-labeled DNA fragments. Catechin and epicatechin induced extensive DNA damage in the presence of Cu(II). Catechin caused piperidine-labile sites at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H₂O₂ and Cu(I). NADH enhanced catechins plus Cu(II)-induced 8-oxodG formation in calf thymus DNA, suggesting the redox cycle between catechins and their corresponding quinones, the oxidized forms of catechins. The DNA-damaging ability of epicatechin is stronger than that of catechin, possibly due to the greater turnover frequency of the redox cycle. The difference in their redox properties could be explained by their redox potentials estimated form an ab initio molecular orbital calculation. The present study demonstrated that catechins could induce metal-dependent H₂O₂ generation during the redox reactions and subsequently damage to cellular and isolated DNA. Therefore, it is reasonably considered that green tea catechins may have the dual function of anticarcinogenic and carcinogenic potentials.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using ³²P-5′-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H₂O₂ generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.     

Catecholamines Enhance Dihydrolipoamide Dehydrogenase Inactivation by the Copper Fenton System. Enzyme Protection by Copper Chelators

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II) /H₂O₂ (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H₂O₂ and CA concentrations. Similar inhibitions were obtained wit! the assayed CAs and o-diphenols. CAs enhanced HO radical production by Cu(II) /H₂O₂, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipo amide, dihydrolipoic acid, DL-dithiothreitol, GSSG, try-panothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H₂O₂/CAs systems. Dena tured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH in creased and Captopril inhibited epinephrine oxidation by Cu(II)/H₂O₂ and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO^* from H₂O₂ by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the: enzyme active site by site-specifically generated HO^* radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his⁺ reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H₂O₂/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H₂O₂/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H₂O₂ formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.     

Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the Fenton Reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical (^?OH) formation from H₂O₂ in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited ^?OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the ^?OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H₂O₂. These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Mechanism of oxidative DNA damage induced by capsaicin,a principal ingredient of hot chili pepper

Although capsaicin exhibits antitumor activity, carcinogenic potential has also been reported. To clarify the mechanism for expression of potential carcinogenicity of capsaicin, we examined DNA damage induced by capsaicin in the presence of metal ion and various kinds of cytochrome P450 (CYP) using ³²P-5′-end-labeled DNA fragments. Capsaicin induced Cu(II)-mediated DNA damage efficiently in the presence of CYP1A2 and partially in the presence of 2D6. CYP1A2-treated capsaicin caused double-base lesions at 5′-TG-3′, 5′-GC-3′ and CG of the 5′-ACG-3′ sequence complementary to codon 273, a hotspot of p53 gene. DNA damage was inhibited by catalase and bathocuproine, a Cu(I) chelator, suggesting that reactive species derived from the reaction of H₂O₂ with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine was significantly increased by CYP1A2-treated capsaicin in the presence of Cu(II). Therefore, we conclude that Cu(II)-mediated oxidative DNA damage by CYP-treated capsaicin seems to be relevant for the expression of its carcinogenicity.     

On the mechanism of the comutagenic effect of Cu(II) with ultraviolet light

Although CuCl₂ alone is not mutagenic inE. coli or in Chinese hamster cells, exposure ofE. coli to CuCl₂ during, UV-irradiation causes enhancement of UV-mutagenesis. The mechanism for this comutagenic effect appears to be owing to increased DNA damage by the combined treatment of UV and Cu(II) compared with UV or Cu(II) alone. Using a sequencing gel approach, UV alone is found to cause a particular pattern of alkali-labile sites, whereas CuCl₂ alone caused few such sites. The combined action of UV+CuCl₂ greatly increased the amount of sites over that of UV alone, and caused a change in their pattern. In the presence of high NaCl concentrations, however, Cu(II) is able to induce DNA damage. This latter effect is most likely owing to the formation of hypochlorite ion. The hypothesis that the comutagenic effect of Cu(II) plus UV might be owing to hydroxyl radical formed via a Fenton reaction involving Cu(II) and UV-generated H₂O₂ was not supported, since no H₂O₂ is detectable in aqueous medium after UV irradiation, and catalase did not block the DNA damage. These results favor the hypothesis that UV-irradiation of Cu(II) causes a photoactivation, enabling it to generate free radicals, perhaps by reacting with dissolved oxygen.     

Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells

Reactive oxygen species threaten genomic integrity by inducing oxidative DNA damage. One common form of oxidative DNA damage is the mutagenic lesion 8-oxoguanine (8-oxodG). One driver of oxidative stress that can induce 8-oxodG is inflammation, which can be initiated by the cytokine tumor necrosis factor alpha (TNF-α). Oxidative DNA damage is primarily repaired by the base excision repair pathway, initiated by glycosylases targeting specific DNA lesions. 8-oxodG is excised by 8-oxoguanine glycosylase 1 (OGG1). A common Ogg1 allelic variant is S326C-Ogg1, prevalent in Asian and Caucasian populations. S326C-Ogg1 is associated with various forms of cancer, and is inactivated by oxidation. However, whether oxidative stress caused by inflammatory cytokines compromises OGG1 variant repair activity remains unknown. We addressed whether TNF-α causes oxidative stress that both induces DNA damage and inactivates S326C-OGG1 via cysteine 326 oxidation. In mouse embryonic fibroblasts, we found that S326C-OGG1 was inactivated only after exposure to H₂O₂ or TNF-α. Treatment with the antioxidant N-acetylcysteine prior to oxidative stress rescued S326C-OGG1 activity, demonstrated by in vitro and cellular repair assays. In contrast, S326C-OGG1 activity was unaffected by potassium bromate, which induces oxidative DNA damage without causing oxidative stress, and presumably cysteine oxidation. This study reveals that Cys326 is vulnerable to oxidation that inactivates S326C-OGG1. Physiologically relevant levels of TNF-α simultaneously induce 8-oxodG and inactivate S326C-OGG1. These results suggest a mechanism that could contribute to increased risk of cancer among S326C-Ogg1 homozygous individuals.     

Relationship between copper biosorption and microbial inhibition of hydroxyl radical formation in a copper(II)–hydrogen peroxide system

The microbial retardation of the spin adduct, DMPO-OH, formed in a copper(II)–hydrogen peroxide–DMPO (5,5-dimethyl-1-pyrroline N-oxide) solution was examined in relation to copper biosorption. A hydroxyl radical is formed in the solution through two steps, the reduction of Cu(II) to Cu(I) by H₂O₂ and the Fenton-type reaction of Cu(I) with H₂O₂. The resultant radical is trapped by DMPO to form DMPO-OH. Microbial cells retarded the DMPO-OH in the Cu(II)–H₂O₂–DMPO far more significantly than in the UV-irradiated H₂O₂–DMPO solution. Egg albumin showed a higher DMPO-OH retardation than microbial cells both in the Cu(II)–H₂O₂–DMPO and the UV-irradiated H₂O₂–DMPO solutions. These results indicated that the retardation effect is related to organic matter and not to microbial activity. Microorganisms having higher affinities for copper ion retarded DMPO-OH more significantly. The linear relationship between the amounts of copper biosorption and the inverse of the median inhibitory doses for DMPO-OH indicated that the microbial cells inhibited the reduction of Cu(II) to Cu(I) by H₂O₂, followed by the decrease of hydroxyl radical formation and the retardation of DMPO-OH. These results also suggest that the coupling between microbial cells and Cu(II) ion can be estimated from their ability to retard DMPO-OH.     

Regulation of a novel cell differentiation-associated gene,JWA during oxidative damage in K562 and MCF-7 cells

Oxidative stress, or the production of oxygen-centered free radicals, has been hypothesized as the major source of DNA damage that can lead to a variety of diseases including cancer. It is known that 8-hydroxy-deoxyguanosine (8-oxo-dG) is a useful biomarker of oxidative DNA damage. Our recent data showed that JWA, initially being cloned as a novel cell differentiation-associated gene, was also actively responsive to environmental stressors, such as heat-shock, oxidative stress and so on. In the present study, we have applied a modified comet assay and bacterial repair endonucleases system (endonuclease III and formamidopyrimidine glycosylase) to investigate if JWA is involved in hydrogen peroxide (H₂O₂)-induced DNA damage and repair in K562 and MCF-7 cells, and to demonstrate if the damage is associated with 8-oxo-dG. The results from the comet assay have shown that the average tail length and the percentage of the cells with DNA tails are greatly induced by H₂O₂ treatment and further significantly enhanced by the post-treatment of repair endonucleases. The H₂O₂-induced 8-oxo-dG formation in K562 and MCF-7 cells is dose-dependent. In addition, the data have clearly demonstrated that JWA gene expression is actively induced by H₂O₂ treatment in K562 and MCF-7 cells. The results suggest that JWA can be regulated by oxidative stress and is actively involved in the signal pathways of oxidative stress in the cells.     

Radical production by hydrogen peroxide/bicarbonate and copper uptake in mammalian cells: modulation by Cu(II) complexes

The presence of the bicarbonate/carbon dioxide pair is known to accelerate the transition metal ion-catalysed oxidation of various biotargets. It has been shown that stable Cu(II) complexes formed with imine ligands that allow redox cycling between Cu(I) and Cu(II) display diverse apoptotic effects on cell cultures. It is also reported that Cu(II)-tetraglycine can form a stable Cu(III) complex. In the present study, radical generation from H₂O₂ and H₂O₂/HCO₃⁻ in the presence of these two different classes of Cu(II) complexes was evaluated by monitoring the oxidation of dihydrorhodamine 123 and NADH and by the quantitative determination of thiobarbituric acid reactive substances (TBARs method). Cu(II)-imine complexes produced low levels of reactive species whereas Cu(II)-Gly-derived complexes, as well as the free Cu(II) ion, produced oxygen-derived radicals in significantly larger amounts. The effects of these two classes of complexes on mammalian tumour cell viability were equally distinct, in that Cu(II)-imine complexes caused apoptosis, entered in cell and remained almost unaffected in high levels whilst, at the same concentrations, Cu(II)-Gly peptide complexes and Cu(II) sulphate stimulated cell proliferation, with the cell managing copper efficiently. Taken together, these results highlight the different biological effects of Cu(II) complexes, some of which have been recently studied as anti-tumour drugs and radical system generators, and also update the effects of reactive oxygen species generation on cell cycle control.     

No significant paraquat-induced oxidative DNA damage in rats

The metabolism of paraquat generates oxygen radicals. Paraquat has thus been suggested as a model compound to induce oxidative damage to DNA, lipids and proteins in different cells and tissues, although experimental data are inconsistent. In order to explore the possibilities for an animal model of oxidative DNA damage in vivo, rats were treated with 20 mg/kg paraquat or vehicle i.p. One and five days later we measured DNA oxidation in terms of 7-hydro-8-oxo-2′-deoxyguanosine (8-oxodG) in the liver and lung as well as the urinary excretion of 8-oxodG. No significant effects on the level of 8-oxodG in the liver, the lung or the urinary excretion, could be distinguished following paraquat treatment. We found, however, a significant correlation (r=0.69; p<0.0002) between the 8-oxodG level in the lung and the urinary excretion, but no significant correlation between the level in the liver and the urinary excretion or between the levels in the liver and the lung. During the experiment the rats were clearly affected by the paraquat as they were very lethargic compared to the controls. Accordingly, even at toxic doses, paraquat did not cause detectable oxidative damage to DNA. The data do not support the use of paraquat as a model compound in experiments investigating effects or prevention of oxidative damage to DNA.     

One-pot synthesis of a new 2-substituted 1,2,3-triazole 1-oxide derivative from dipyridyl ketone and isonitrosoacetophenone hydrazone: Nickel(II) complex,DNA binding and cleavage properties

An efficient and simple one-pot synthesis of a new 1,2,3-triazole-1-oxide via reaction between isonitrosoacetophenone hydrazone and dipyridyl ketone in the EtOH/AcOH at room temperature has been developed smoothly in high yield. The reaction proceeds via metal salt free, in-situ formation of asymmetric azine followed by cyclization to provide 1,2,3-triazole 1-oxide compound. It has been structurally characterized. The 1:1 ratio reaction of the 1,2,3-triazole 1-oxide ligand with nickel(II) chloride gives the mononuclear complex [Ni(L)(DMF)Cl₂], hexa-coordinated within an octahedral geometry. Characterization of the 1,2,3-triazole compound and its Ni(II) complex with FTIR, ¹H and ¹³C NMR, UV–vis and elemental analysis also confirms the proposed structures of the compounds. The interactions of the compounds with Calf thymus DNA (CT-DNA) have been investigated by UV–visible spectra and viscosity measurements. The results suggested that both ligand and Ni(II) complex bind to DNA in electrostatic interaction and/or groove binding, also with a slight partial intercalation in the case of ligand. DNA cleavage experiments have been also investigated by agarose gel electrophoresis in the presence and absence of an oxidative agent (H₂O₂). Both 1,2,3-triazole 1-oxide ligand and its nickel(II) complex show nuclease activity in the presence of hydrogen peroxide. DNA binding and cleavage affinities of the 1,2,3-triazole 1-oxide ligand is stronger than that of the Ni(II) complex.     

l-Histidyl-glycyl-glycyl-l-histidine. Amino-acid structuring of the bleomycin-type pentadentate metal-binding environment capable of efficient double-strand cleavage of plasmid DNA

A tetrapeptide, l-histidyl-glycyl-glycyl-l-histidine (HGGH), was synthesized and the pUC19 plasmid DNA cleaving activity by copper(II) complex of HGGH (Cu(II)−HGGH) was investigated. Cu(II)−HGGH showed bleomycin-like DNA cleaving activity and, at 50 nM, converted a supercoiled DNA efficiently to a linear DNA in the presence of 500 μM H₂O₂/sodium ascorbate through an oxidative pathway.     

Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using ³²P-labeled human DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5′-GG-3′, 5-GGGG-3′, 5′-GGGGG-3′ sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H₂O₂ and Cu(I). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could beproduced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.